Massive bone and joint defects arise from resection of primary and metastatic tumors, congenital deficiency, traumatic loss, infection, or prosthetic implant failure. Available reconstructive methods are prone to high rates of failure. They include structural allografts (infection, nonunion and stress fracture), vascularized autografts (limited availability, size/shape mismatch, morbidity), or prosthetic replacement (infection, periprosthetic fracture and failure). A better solution to this difficult problem is required. Transplantation of living allogenic bone/joint would allow close matching of defect dimension and structure, while simultaneously maintaining the functional and healing properties of living tissue. Long-term immune modulation is necessary at present, unacceptable due to substantial health risks of immunosuppressive drugs or tolerance induction for such non- life critical transplantation. Maintaining allogeneic tissue viability without immunosuppression would be an important advance, and is the goal of this grant renewal proposal. Therapeutic angiogenesis is used to develop a host-derived neoangiogenic circulation within the transplanted bone/joint that maintains blood flow regardless of immune status. Previous work has demonstrated the method's promise. The Specific Aims of this proposal are #1) to test whether local delivery of vasculogenic growth factors improve measures of capillary formation and bone blood flow in living femoral allotransplants with simultaneous recipient AV bundle implantation, #2) to determine whether local delivery of a bone morphogenic protein may enhance new bone formation in the same model, #3 )to investigate whether osteocytes in newly formed bone are of recipient rather than transplant origin, and #4) to evaluate the viability, healing potential and function of orthotopic whole joint composite tissue transplants in a larger animal (rabbit knee) model. Improved clinical outcome of limb salvage surgery performed in difficult circumstances is the ultimate aim of this basic research. Methods: In aims 1, 2 and 3, sex-mismatched vascularized femora are transplanted using a saphenous arteriovenous (AV) bundle to provide host-derived angiogenesis. Growth factors are delivered, encapsulated within biodegradable microspheres placed within the transplanted femur. Angiogenesis is measured at two time points by quantifying capillary density with microangiography and bone blood flow by hydrogen washout;while bone remodeling is determined after fluorochrome labeling by quantitative histomorphometry. Laser capture microdissection will be used to study lineage of osteocytes specifically located in areas of newly formed bone, as measured by quantitative real-time PCR for the Y-chromosome-specific SRY gene (thus defining host or graft origin of the cells). Following whole knee joint transplants in a rabbit model, mechanical properties of joint cartilage and kinematic analysis of joint function will be correlated with measures of blood flow, angiogenesis and histology to assess the method's use in composite tissue allotransplantation. PUBLIC HEALTH RELEVANCE: Segmental bone defects are commonly encountered in orthopedic practice but available reconstructive methods, including structural allografts, vascularized autografts and prosthetic replacement are prone to high rates of failure. Transplantation of living allogenic bone and/or joint is a potential solution that would allow close matching of defect dimension and structure, while simultaneously maintaining the functional and healing properties of living tissue. The goal of this grant renewal proposal is to study the means, mechanism of action and effectiveness of a surgically-created host-derived neoangiogenic circulation to maintain blood flow and function in living musculoskeletal tissue transplants without need for prolonged postoperative drug therapy or tolerance induction.